PROJECT SUMMARY The worldwide adoption of artemisinin-based combination therapies (ACTs) has been instrumental in halving the global burden of Plasmodium falciparum (Pf) malaria since the early 2000s. Malaria?s impact nonetheless remains vast, with over 400,000 yearly deaths in Africa alone. Now, Pf resistance to ACTs threatens to overwhelm malaria control efforts. Treatment failure rates with the ACT dihydroartemisinin+piperaquine (PPQ) currently exceed 50% in Cambodia, and resistance to other ACTs has been observed elsewhere in Asia. The emergence and spread of ACT resistance in Africa would be calamitous. Studies have identified core roles for the Pf chloroquine resistance transporter PfCRT and the multidrug resistance transporter PfMDR1 in modulating Pf susceptibility to the ACT partner drugs PPQ, amodiaquine (ADQ), pyronaridine (PND), lumefantrine (LMF) and mefloquine (MFQ). These studies include our recent gene editing-based finding that novel PfCRT mutations present in Cambodia can confer PPQ resistance. By analyzing a dataset of >2,500 Pf genomes sampled across Asia and Africa, we now find a plethora of new sequence variants of both genes. In Aim 1, we will apply gene editing techniques coupled with comprehensive drug susceptibility profiling to test the hypothesis that these novel PfCRT mutations have been selected to mediate ACT partner drug resistance. In Asia, we predict that these variants primarily affect susceptibility to PPQ, LMF, or MFQ. In Africa, we observe a predominance of previously uncharacterized haplotypes, and will characterize whether these variants mediate reduced susceptibility to partner drugs and/or minimize fitness costs. In Aim 2, we will test the hypothesis that current ACTs are selecting for novel mutations in PfMDR1 throughout Asia and Africa. Based on our recent discovery of a role for PfMDR1 as a mediator of gametocyte resistance to antimalarials, we will also assess whether mutant PfMDR1 isoforms can enhance the transmission of drug-resistant Pf parasites. In Aim 3, we propose to identify antimalarial combinations that exert opposing selective pressures on PfCRT and PfMDR1 and thus preclude the acquisition of multidrug resistance. Using selection methods with mutant pfcrt and pfmdr1 lines, our studies will experimentally address the premise of two ongoing triple ACT clinical trials in Cambodia, which are testing the dual partner drug combinations LMF+ADQ and PPQ+MFQ as approaches to effectively treat drug-resistant malaria and prevent its further evolution and spread. In Aim 4, we will use cell- based assays to test the hypothesis that hemoglobin endocytosis and subsequent processing in the Pf digestive vacuole are key aspects of the modes of action of most ACT drugs, whose potency can be impacted by mutant isoforms of PfMDR1 or PfCRT. This proposal, which aligns with the NIAID priority of supporting research on antimicrobial drug resistance, will transform our understanding of ACT partner drug resistance and modes of action, provide new biomarkers, and identify strategies to effectively treat drug-resistant Pf malaria.